Near-infrared cyanine dyes and conjugates thereof

ABSTRACT

The present invention relates to the field of optical imaging. More particularly, it relates to compounds of the cyanine family with near-infrared emission characterized by improved physico-chemical and biological properties and to conjugates with biological ligands thereof. The invention also relates to the use of these compounds as optical diagnostic agents in imaging or therapy of solid tumors, to the methods for their preparation and to the compositions comprising them. The compounds have formula (I), formula (I), wherein X is direct bond or —O—; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl, substituted by at least two hydroxyl groups; R1 and R2 are each independently a linear or branched C1-C6 alkyl substituted by a group selected from —SO3H, —COOH, —CONH2 and —COO—C1-C6 alkyl; and R3 is hydrogen, —SO3H or a linear or branched C1-C6 alkyl substituted by —COOH or —CONH—Y, wherein Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclyl, substituted by at least two hydroxyl groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage application of correspondinginternational application number PCT/EP2021/066920, filed Jun. 22, 2021,which claims priority to and the benefit of European application no.20181771.5, filed Jun. 23, 2020.

FIELD OF THE INVENTION

The present invention relates to the field of optical imaging. Moreparticularly, it relates to compounds of the cyanine family withnear-infrared emission characterized by improved physico-chemical andbiological properties and to conjugates with biological ligands thereof.The invention also relates to the use of these compounds as opticaldiagnostic agents in imaging or therapy of solid tumors, to the methodsfor their preparation and to the compositions comprising them.

BACKGROUND ART

Dyes are chemical entities that absorb photons of a specific wavelengthupon light excitation and re-emit some of that energy, depending onquantum efficiency, usually at a longer wavelength. Particularly,cyanine dyes are fluorescent organic molecules characterized by adelocalized electron system that spans over a polymethine bridge and isconfined between two nitrogen atoms. Some of them, having favourableoptical properties, low toxicity and good solubility in aqueous media,can be used as contrast agents for biomedical imaging. Cyanine dyesemitting in the near-infrared region (700-900 nm) are particularlyuseful for biomedical imaging applications due to the higher penetrationdepth compared to dyes with fluorescence emission in the visiblespectrum.

Among the near-infrared dyes used for biomedical imaging, Indocyaninegreen (ICG) is the only medicinal product currently approved for humanuse. ICG is routinely used to assess tissue perfusion and forangiographic applications due to the strong binding to plasma protein(blood pool effect) and rapid clearance of the unbound fraction by theliver (Cherrick et al., J Clin Invest 1960; 39(4): 592-600).Furthermore, ICG is also tested as investigational medicinal product fortumor imaging during diagnostic and interventional (fluorescence-guidedsurgery) procedures. ICG distributes and accumulates in tumor tissues bya combination of passive diffusion and enhanced permeability andretention (EPR) effect (Onda N. et al., Int J Cancer 2016; 139,673-682). False positives are common clinical findings associated withthe use of ICG for tumor imaging due to the non-specific accumulationproperties (Tummers Q. et al., PlosOne 2015; 10(6): e0129766).

Further contrast agents for near-infrared imaging are under developmentwhich exploit the use of a dye conjugated to a carrier moiety (i.e.,biomolecule), targeting an overexpressed tumor epitope, to improvesensitivity and specificity of detection (Achilefu S. et al, J Med Chem2002; 45, 2003-2015). For instance, ICG and S0456 are examples ofnear-infrared dyes that have been conjugated to tumor-targeting moietiesand are currently tested in clinical trials for intraoperative tumordetection (Fidel J. et al., Cancer Res. 2015; 15; 75(20): 4283-4291;Hogstins C. et al., Clin Cancer Res 2016; 22(12); 2929-38).

Despite several efforts to find suitable imaging agents, there is stillthe need to find improved dyes endowed with optimal solubility and lowaggregation in aqeous media, high fluorescence efficiency and optimalbiological properties. The biological properties of the dye, especiallythe binding affinity to plasma proteins such as albumin, may stronglyimpact distribution and tissue accumulation once administered into aliving organism. For instance, dyes with high binding affinity to humanalbumin are sequestered in the plasma compartment after intravenousadministration, and have low tissue extravasation rate which stronglylimits their diagnostic applications. Furthermore, the biologicalproperties of the dye may influence the tissue distribution ofconjugates composed of the dye itself and a biomolecule targeting abiological epitope on a pathological tissue. Near-infrared dyes endowedwith with low binding affinity for human serum albumin and non-specificaccumulation would be preferable for applications in living organisms.This need is paramount when the dye is conjugated to a biomolecule thatspecifically binds a molecular epitope or a pathologic tissue (e.g. atumor). The present invention addresses these and other needs.

WO2002/024815 and WO2007/136996 in the name of Li-Cor Inc. andWO2004/065491 in the name of Schering AG report stable cyanine dyesuseful for optical imaging applications and characterized by highsolubility in aqueous media and functional groups for direct conjugationwith biomolecules. However, no teachings are therein provided about howto obtain dyes with optimal biological properties.

WO2015/114171 discloses small molecule targeted drug conjugates fordelivery of drugs to inhibit the cancer cells. In particular, it reportsthe IRDye 750 conjugate “C6” used for flow cytometry analisis and invivo imaging of tumors.

Wada H. et al, Chemical Engineering Journal 2018, 340 (3): 51-57discloses NIR fluorescent nanoprobes using mannose-conjugated ZW800-1derivatives for intraoperative pan lymph nodes mapping and real-timeoptical imaging.

Vendrell M. et al, Organic & Biomolecular Chemistry 2011,9 (13):4760-4762 reports a NIR fluorescent deoxyglucose analogue CyNE 2-DGwhich showed a preferential uptake in cancer cells and was validated asoptical agent in imaging of tumors.

Despite several efforts to find suitable imaging agents, there is stillthe need to find improved dyes endowed with optimal stability andfluorescence efficiency, as well as optimal physicochemical andbiological properties, and designed for optical imaging of livingorganisms. This need is paramount particularly when the dye isconjugated to a biomolecule that specifically binds a molecular epitopeor a pathologic tissue (e.g. a tumor). The present invention addressesthese and other needs.

SUMMARY OF THE INVENTION

Generally, object of the present invention is to provide new cyaninedyes, or their corresponding conjugates to binding moieties, useful ascontrast medium for optical imaging and aimed at solving the abovementioned issues.

The new cyanine derivatives described herein are surprisingly endowedwith remarkable optical properties and high solubility in aqeous media.Surprisingly, it has been found that the compounds of the invention havea very low binding affinity for human albumin compared to near-infrareddyes known in the prior art, which is particularly advantageous whenthese compounds are used after intravenous administration; said lowaffinity prevents the sequestration of the compounds in the plasmacompartment by the large proteins present in the blood, such as albumin,and the consequent reduction of the fraction of free dye available forefficient extravasation and distribution in the extracellular space.

The new cyanine dyes can be conveniently conjugated to suitabletargeting moieties through suitable functional groups acting as bindingsites, thus providing very specific and sensitive contrast agents formolecular imaging. This low albumin binding affinity of the compounds ofthe invention is particularly important in case of dyes-conjugates,since only their free fraction (not bound to albumin) can efficientlyinteract with the molecular target.

A further aspect of the invention relates to such dyes as diagnosticagents, in particular for use in optical imaging of a human or animalorgan or tissue, for use in a method of optical imaging, wherein theimaging is a tomographic imaging of organs, monitoring of organfunctions including angiography, tissue perfusion imaging, urinary tractimaging, bile duct imaging, nerve imaging, intraoperative canceridentification, fluorescence-guided surgery, fluorescence endoscopy,fluorescence laparoscopy, robotic surgery, open field surgery, laserguided surgery, photodynamic therapy, fluorescence lifetime imaging, ora photoacoustic or sonofluorescence method.

Moreover the invention relates to a manufacturing process for thepreparation of the provided dyes, the corresponding conjugates and/orthe pharmaceutically acceptable salts thereof, and to their use in thepreparation of a diagnostic agent.

According to a further aspect, the invention relates to apharmaceutically acceptable composition comprising at least one dye ordye-conjugate compound of the invention, or a pharmaceuticallyacceptable salt thereof, in a mixture with one or more physiologicallyacceptable carriers or excipients. Said compositions are useful inparticular as optical imaging agents to provide useful imaging of humanor animal organs or tissues.

In another aspect, the present invention refers to a method for theoptical imaging of a body organ, tissue or region by use of an opticalimaging technique that comprises the use of an effective dose of acompound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is a first object of the present invention the provisionof a compound of formula (I),

wherein

-   -   X is direct bond or —O—;    -   Y is a group selected from linear or branched C₁-C₆ alkyl, C₃-C₇        cycloalkyl and heterocyclyl, substituted by at least two        hydroxyl groups;    -   R1 and R2 are each independently a linear or branched C₁-C₆        alkyl substituted by a group selected from —SO₃H, —COOH, —CONH₂        and —COO—C₁-C₆ alkyl; and    -   R3 is hydrogen, —SO₃H or a linear or branched C₁-C₆ alkyl        substituted by —COOH or —CONH—Y, wherein Y is a group selected        from linear or branched C₁-C₆ alkyl, C₃-C₇ cycloalkyl and        heterocyclyl, substituted by at least two hydroxyl groups,

or a stereoisomer or pharmaceutically acceptable salt thereof.

Another object of the present invention relates to the correspondingconjugated dyes represented by a compound of formula (II)

wherein

-   -   X is direct bond or —O—;    -   Y is a group selected from linear or branched C₁-C₆ alkyl, C₃-C₇        cycloalkyl and heterocyclyl, substituted by at least two        hydroxyl groups;    -   R1 is linear or branched C₁-C₆ alkyl substituted by a group        selected from —SO₃H, —COOH, —CONH₂ and —COO—C₁-C₆ alkyl;    -   R4 is linear or branched C₁-C₆ alkyl substituted by a group        selected from —SO₃H, —COOH and —CONH—(S)_(m)-T, wherein    -   S is a spacer;    -   T is a targeting moiety; and    -   m is an integer equal to 0 or 1; and    -   R5 is selected from hydrogen, —SO₃H, a linear or branched C₁-C₆        alkyl, substituted by —COOH or —CONH—Y, and a group        CONH—(S)_(m)-T, wherein Y, S, T and m are defined above;        and wherein at least one between R4 and R5 is linear or branched        C₁-C₆ alkyl substituted by CONH—(S)_(m)-T,        or a stereoisomer or pharmaceutically acceptable salt thereof.

The present invention also relates to methods for preparing thecompounds of formula (I) or (II) by means of synthetic transformationssteps.

The invention also comprises compounds of formula (I) or (II) for use asfluorescent probes for biomedical optical imaging applications.

Definitions

In the present description, and unless otherwise provided, the followingterms and phrases as used herein are intended to have the followingmeanings.

The expression “straight or branched C₁-C₆ alkyl” refers to an aliphatichydrocarbon radical group, which may be a straight or branched-chain,having from 1 to 6 carbon atoms in the chain. For instance, “C₄ alkyl”comprises within its meaning a linear or branched chain comprising 4carbon atoms. Similarly, “C₁-C₂₀ alkyl” is an alkyl comprising from 1 to20 carbon atoms. Representative and preferred alkyl groups includemethyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, pentyl andhexyl. Unless otherwise specified, the straight or branched C₁-C₆ alkylis a monovalent radical group. In some cases it may be a “bivalent” or“multivalent” radical group, wherein two or more hydrogen atoms areremoved from the above hydrocarbon radical group and substituted, e.g.methylene, ethylene, iso-propylene groups and the like.

The term “C₃-C₇ cycloalkyl” as used therein comprises within its meaninga saturated (i.e. cycloaliphatic) carbocyclic ring comprising from 3 to7 carbon atoms. Suitable examples include a C₅-C₇ carbocyclic ring, e.g.a cyclohexyl ring.

The term “heterocyclyl” as used therein comprises a saturatedcycloaliphatic ring, preferably a 5-7 membered saturated ring, furthercomprising an heteroatom in the cyclic chain selected from N, O and S.Preferably, it refers to tetrahydropyran.

The term “hydroxyalkyl” refers to any of the corresponding alkyl chainwherein one or more hydrogen atoms are replaced by hydroxyl groups.

The term “alkoxy” comprises within its meaning an alkyl chain as abovedefined further comprising one or more oxygen atoms; examples include,for instance, alkyl-oxy groups such as methoxy, ethoxy, n-propoxy,iso-propoxy and the like, and alkyl-(poly)oxy groups in which the alkylchain is interrupted by one or more oxygen atoms.

In the present description the term “protecting group” (Pg) designates aprotective group adapted for preserving the function of the group towhich it is bound. Specifically, protective groups are used to preserveamino, hydroxyl or carboxyl functions. Appropriate protective groups mayinclude, for example, benzyl, carbonyl, such as formyl,9-fluoromethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz),t-butoxycarbonyl (Boc), isopropyloxycarbonyl or allyloxycarbonyl(Alloc), alkyl, e.g. tert-butyl or triphenylmethyl, sulfonyl, acetylgroups, such as trifluoroacetyl, benzyl esters, allyl, or othersubstituents commonly used for protection of such functions, which arewell known to the person skilled in the art (see, for instance, thegeneral reference T. W. Green and P. G. M. Wuts, Protective Groups inOrganic Synthesis, Wiley, N.Y. 2007, 4th Ed., Ch. 5).

Moreover, the invention comprises also the precursors or intermediatescompounds suitable for the preparation of a desired compound of formula(I) or salts thereof. In such derivatives the functional groups ofR1-R5, such as a carboxylic acid or carboxamide, can be protected withan appropriate protecting group (Pg) as defined above, preferably withalkyl or ester groups. If necessary, also hydroxyl groups of Y groupscan be protected with an appropriate protecting group (Pg) during thepreparation of the compounds of formula (I) or (II), thus forming forinstance acetoxy, alkoxy or ester groups.

The expression “coupling reagent” refers to a reagent used for instancein the formation of an amide bond between a carboxyl moiety and an aminomoiety. The reaction may consist of two consecutive steps: activation ofthe carboxyl moiety and then acylation of the amino group with theactivated carboxylic acid. Non limiting examples of such coupling agentsare selected from the group consisting of: carbodiimides, such asN,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC); phosphoniumreagents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP), 7-azabenzotriazol-1-yloxy-tripyrrolidino-phosphoniumhexafluorophosphate (PyAOP), [ethyl cyano(hydroxyimino)acetato-C2]tri-1-pyrrolidinylphosphonium hexafluorophosphate(PyOxim), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP)and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT); andaminium/uronium-imonium reagents, such asN,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate(TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (H BTU),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU),O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HCTU),1-[1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uroniumhexafluorophosphate (COMU) and fluoro-N,N,N′,N′-tetramethylformamidiniumhexafluorophosphate (TFFH) or other compounds well known to the personskilled in the art.

The terms “small molecule” is broadly used therein to refer to anorganic, inorganic or organometallic compound having a molecular weightof less than about 5000 Daltons and being able to regulating abiological process or causing a biological effect when administered toan animal, including humans. The term “small molecule” can be also usedinterchangeably with the terms “drug” or “biologically active moiety”.For instance, it can include any agent, active molecule or compoundwhich provides a beneficial effect against tumors and produces alocalized or systemic effect in a patient by binding a specificbiological target. A small molecule can also refer to a portion orresidue of a parent drug, which is activated or chemically modified fromthereof and covalently attached to a conjugated dye of the invention.

The expression “activated carboxylic acid” refers to a derivative of acarboxyl group that is more susceptible to nucleophilic attack than afree carboxyl group; suitable derivatives may include for instance acidanhydrides, thioesters, acyl halides, NHS ester and sulfo NHS esters.

Moreover, the terms “moiety” or “residue” are herewith intended todefine the residual portion of a given molecule once properly attachedor conjugated, either directly or through a suitable linker and/orspacer, to the rest of the molecule.

Targeting moiety (T)

According to the invention, a targeting moiety (T) is a molecule thatbinds with particular selectivity to a biological target and facilitatesthe accumulation of the contrast agent in a specific tissue or part ofthe body. Generally, it is represented by a natural or syntheticmolecule for use in biological systems.

Such specific binding can be achieved through a ligand, such as forinstance a small molecule, a protein, a peptide, a peptidomimetic, anenzyme substrate, an antibody or fragment thereof or an aptamer,interacting with a specific biological target expressed on the surfaceof the tissues or cells of interest.

Suitable biological targets for the compounds of the invention can befor instance an epithelial growth factor (EGF) receptor, such as EGFR orHER2; a vascular endothelial growth factor (VEGF) receptor, such asVEGFR1 or VEGFR2; a carbonic anhydrase (CA) enzyme, such as CAIX, CAIIor CAXII; a mucin glycoprotein, such as MUC1; a glucose transporter,such as GLUT-1; a sodium-hydrogen antiporter, such as NHE1; acarcinoembryonic glycoprotein, such as the carcinoembryonic antigen(CEA); a chemokine receptor, such as the chemokine receptor type 4(CXCR4); a cell adhesion molecule, such as ICAM, EPCAM, VCAM,E-Selectin, P-Selectin; the hepatocyte growth factor HGFR (c-met); areceptor for the transferrin; a ephrin receptor, such as EPHA2; areceptor for the folic acid, such as FR-alpha; a glycoprotein bindingialuronic acid, such as CD44; a bombesin receptor, such as BB1, BB2,BB3; a N-acetyl-L-aspartyl-L-glutamate (NAAG) peptidase, such asprostate-specific membrane antigen (PSMA); and, in particular, anintegrin receptor, such as α_(v)β₃, α_(v)β₅, α_(v)β₆ or α₅β₁ integrinreceptors.

For instance, integrin receptors targeting moieties are represented bylinear or cyclic peptides comprising the sequence Arg-Gly-Asp (RGD).This tripeptide has high binding specificity for the receptor, beingrecognized as ligand by the family of the integrin receptors located inthe cell membrane. In fact, it has been identified in some extracellularmatrix glycoproteins, such as fibronectin or vitronectin, which exploitthis RGD motif to mediate cell adhesion.

Therefore, linear and cyclic peptides and peptidomimetics containing thesequence Arg-Gly-Asp (RGD), such as for instance cRGD, cRGDfK, cRGDyK,cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., SciRep, 2017, 7:3905), or their analogues and derivatives thereof, are awell known example of binding motif targeting cancer tissues on whichcell membrane integrins are up-regulated compared to healthy tissues.

In one embodiment, the compounds of the invention can be conjugated tovectors known to target the prostate-specific membrane antigen (PSMA),thus allowing the detection and imaging of prostate cancer. Theseligands are represented for instance by the vector glutamicacid-urea-lysine (EuK) or other PSMA binding vectors of formula “EuX” asdescribed in EP3636635 A1, namely glutamic acid linked to another aminoacid or similar via a bridging urea, for example EuFA (glutamicacid-urea-3-(2-furyl)-alanine), EuPG (glutamicacid-urea-2-(2′-propynyl)-alanine), EuE (glutamic acid-urea-glutamicacid), or other urea-based peptidomimetics, such as for instanceEuK-(3-(2-naphtyl)-alanine)-tranexamic acid, as described in Benešová etal., J Nucl Med 2015, 56:914-920.

In another embodiment, the compounds of the invention can be conjugatedto other small molecules, peptides, proteins or antibodies, such as forinstance monoclonal antibodies already used for therapy. Small moleculescontaining the drug acetazolamide, such as for instance compounds 4a,5a, 6a, 7a and 8a (Wichert et al., Nat Chem 2015, 7:241-249), or theiranalogues and derivatives thereof, are examples of small moleculestargeting the enzyme CAIX. Linear and cyclic peptides andpeptidomimetics, such as peptide GE11 (described in Li et al., FASEB J2005, 19:1978-85) and/or peptide L1 (described in Williams et al., ChemBiol Drug Des 2018, 91:605-619), or their analogues and derivativesthereof, are examples of peptides targeting the epithelial growth factorreceptor (EGFR). Among the proteins, derivatives of the epithelialgrowth factor (EGF) are examples of small protein targeting theepithelial growth factor receptor (EGFR). Among the antibodies,panitumumab and cetuximab are examples of monoclonal antibodiestargeting the epithelial growth factor receptor (EGFR).

Preferably, the targeting ligands of the invention are able toselectively link tumor cells or tissues. In particular they are able tolink tumors selected from brain cancer, breast cancer, head and neckcancer, ovarian cancer, prostate cancer, esophageal cancer, skin cancer,gastric cancer, pancreatic cancer, bladder cancer, oral cancer, lungcancer, renal cancer, uterine cancer, thyroid cancer, liver cancer, andcolorectal cancer. In addition, the targeting ligands are able to linkmetastatic spreads of the above-mentioned cancers in tissues and organsdifferent from the primary source. Furthermore, the targeting ligandsare able to link pre-neoplastic lesions and dysplasia in differenttissues and organs.

Spacer S

According to the invention, S is a spacer, optionally present, thatseparates the targeting moiety from the dye. The presence of a spacer isparticularly relevant for some embodiments where the targeting moietyand the dye risk to adversely interact with each other. Moreover, thepresence of the spacer may be necessary when the dye is relatively largeand may interfere with the binding of the targeting moiety to the targetsite.

The spacer can be either flexible (e.g., simple alkyl chains) or rigid(e.g., cycloalkyl or aryl chains) so that the dye is oriented away fromthe target. The spacer can also modify pharmacokinetic and metabolism ofthe conjugates of formula (I) used as imaging agents in a livingorganism.

Hydrophilic spacers may reduce the interaction with plasma proteins,reduce blood circulation time and facilitate excretion. For example, ifthe spacer is a polyethyleneglycol (PEG) moiety, the pharmacokineticsand blood clearance rates of the imaging agent in vivo may be altered.In such embodiments, the spacer can improve the clearance of the imagingagent from background tissue (i.e., muscle, blood) thus giving a betterdiagnostic image due to high target-to-background contrast. Moreover,the introduction of a particular hydrophilic spacer may shift theelimination of the contrast agent from hepatic to renal, thus reducingoverall body retention.

Therefore, in one preferred embodiment, the spacer is an hydrophilicmoiety comprising C₁-C₂₀ alkyl, C₃-C₇ cycloalkyl or aryl groups.Preferably, the spacer is selected from the group consisting of—(CH₂)_(p)COO—, —(CH₂CH₂O)_(p)CH₂CH₂COO— and —(CH₂CH₂O)_(p)CH₂CH₂NH—,wherein ρ is an integer between 0 and 20. Preferably ρ is 2, 6 or 12.

When not necessary, the spacer is preferably absent, i.e. m is 0 and Srepresents a direct bond. The spacer, or alternatively the targetingmoiety when the spacer is absent, can be connected in a compound offormula (II), alternatively at the R4 and/or R5 residue.

The linking groups of R4-R5 are reactive functional groups such ascarboxylic acid or carboxamido residues suitable for conjugating the dyeto the targeting moiety by formation of a chemical bond. For instance,when an amine-containing targeting moiety (T) is conjugated with acompound of formula (II) wherein R4 and/or R5 is an alkyl substituted bycarboxylic acid, this carboxylic acid may be optionally activated beforecarrying out the conjugation through conversion in a more reactive formusing an activating reagent, forming for example a N-hydroxy succinimide(NHS) ester or a mixed anhydride. Then, to obtain the correspondingcompound of formula (II), the amine-containing targeting moiety istreated with the resulting activated acid to form an amide linkage.Typically, this reaction is carried out in aqueous buffer, optionalco-solvent with DMSO or DMF at pH 8 to 9, or in organic solvent withorganic bases such as DIPEA, TEA or NMM.

Otherwise a direct conjugation using the “non-activated” carboxylic acidmay be performed.

Similarly, when the linking group of R4 and/or R5 is a carboxamidogroup, the procedure for attachment of the suitable targeting moiety isanalogous, but no activation step of the linker is generally requiredand the dye and targeting moiety are treated directly.

The compounds of the above formula (I) or (II) may have one or moreasymmetric carbon atoms, otherwise referred to as chiral carbon atoms,and may thus give rise to diastereomers and optical isomers. Unlessotherwise provided, the present invention further includes all suchpossible diastereomers as well as their racemic mixtures, theirsubstantially pure resolved enantiomers, all possible geometric isomers,and pharmaceutically acceptable salts thereof.

As a non limiting example, when the dyes of formula (I) or (II) aresubstituted by a D-glucamine group (Y is 06 alkyl substituted with fivehydroxy groups), the invention comprises the correspondingenantiomerically pure compounds as well as any stereoisomers thereof,for instance compounds bearing a L-glucamine group or any possiblemixtures of D-/L-enantiomers thereof. The present invention furtherrelates to compounds of the above formula (I) or (II) in which thefunctional groups of R1, R2/R4 and/or R3/R5, e.g. the sulfonyl,carboxyamino or carboxylic acid groups, may be in the form of apharmaceutically acceptable salt.

In one embodiment, the invention relates to a compound of formula (I) or(II) wherein Y is selected from a linear or branched C₁-C₆ alkyl,cycloalkyl and heterocyclyl, substituted with from two to five hydroxylgroups.

In a preferred embodiment the invention relates to a compound of formula(I) or (II) wherein Y is selected from the group consisting of

More preferably, the invention relates to a compound of formula (I) or(II) wherein Y is a group of formula (ii) as defined above. Preferably,the group (ii) has the following stereochemical configuration obtainedby using a D-glucamine in the preparation of the compounds:

Another embodiment of the invention relates to a compound of formula(II) wherein m is 0 and the spacer S is represented by a direct bond orm is 1 and the spacer is an hydrophilic moiety comprising C₁-C₂₀ alkyl,C₃-C₇ cycloalkyl or aryl groups. Preferably, the spacer is selected from—(CH₂)_(p)COO—, —(CH₂CH₂O)_(p)CH₂CH₂COO— and —(CH₂CH₂O)_(p)CH₂CH₂NH—,wherein ρ is an integer between 0 and 20. Preferably ρ is 2, 6 or 12.

In a further embodiment, T is a targeting moiety selected from a smallmolecule, a protein, a peptide, a peptidomimetic, an enzyme substrate,an antibody or any fragment thereof and an aptamer.

Preferably T is represented by a peptide, and in particular by a moietyinteracting with an integrin receptor, such as α_(v)β₃, α_(v)β₅,α_(v)β₆, α₅β₁ and the like, preferably with α_(v)β₃ integrin receptor.In a preferred embodiment, R1 is a linear C₄ alkyl substituted by —SO₃H.In another preferred embodiment, the invention relates to a compound offormula (I) or (II) wherein Y represents a group (ii) as defined above,otherwise represented by the following formula (Ia) or (Ila)respectively:

wherein R1, R2, R3, R4, R5 and X are as defined above.

Especially preferred are the compounds of formula (I) listed in Table 1a and the compounds of formula (II) listed in Table Ib.

TABLE la Preferred compounds of formula (I)

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

TABLE Ib Preferred conjugated dyes of formula (II)

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

The present invention is also directed to methods for synthesizing thecompounds of formula (I) or (II), prepared as illustrated in thefollowing of the description, which are near-infrared dyes optionallyconjugated to a targeting moiety through a linking group.

Accordingly, the invention provides the compounds of formula (I) or (II)as defined above for use as optical imaging agents for diagnosticbiomedical applications in mammals (humans and animals). Preferably theimaged mammal subject is a human.

In a preferred embodiment, the compounds of the invention are for use asimaging agents in the detection of normal (healthy) tissues or abnormal(pathologic) tissues, in particular a tumor.

Preferably, the compounds of formula (I) or (II) as defined above arefor use in the detection of normal (healthy) tissues by means of imagingtechniques comprising for instance angiography, perfusion imaging, bileduct imaging and nerve imaging.

In a further preferred embodiment, the invention provides for compoundsof formula (I) or (II) as defined above for use in the detection ofabnormal (pathologic) tissue such as for instance a primary tumorlesion, local or distant metastases, or a pre-neoplastic lesion, inparticular dysplasia and hyperplasia. In particular, the compounds offormula (II) as defined above are preferably for use in the detectionand demarcation of a tumor margin in guided surgery of an individualpatient. A preferred use is wherein said tumor is a tumor showing aover-expression of a biological epitope, for instance selected from areceptor, an enzyme, a glycoprotein, a lipid raft, a transmembraneprotein located on the cell surface and a soluble factor present inserum, plasma or the interstitial space. Preferably, said biologicalepitope is an integrin receptor for Vitronectin, Fibrogen and/or for thetransforming growth factor-β (TGF-β). The invention also provides acompound of formula (I) or (II) for use as fluorescent probe as definedabove, wherein the detection and demarcation of the tumor is carried outunder NIR radiation. Preferably, such detection and demarcation of tumoris carried out before, during or after a surgical procedure to removesuch tumor tissue. A fluorescence-guided surgery procedure is an exampleof such use.

Additionally, the invention provides compounds of formula (I) or (II) asdefined above for use in the detection of an inflamed tissue, a fibrotictissue, an ischemic tissue, or a tissue with abnormal metabolic rate.

The invention also provides for a method of imaging tissues and cellscomprising the steps of:

-   -   i) contacting the cells or tissues with a compound of        formula (I) and (II);    -   ii) irradiating the tissues or cells at a wavelength absorbed by        the imaging agent;    -   iii) detecting the near-infrared emission using a fluorescence        camera.

Preferably, said contacting the cells or tissues with the imaging agentsof formula (I) or (II) is accomplished by topical or local application(e.g., by spraying, soaking or applying an ointment, foam or cream) orby systemic application (enteral or parenteral administration).

The invention further relates to a pharmaceutical diagnostic compositioncomprising a compound of formula (I) or a conjugate of formula (II) asdefined above, and at least one pharmaceutically acceptable carrier orexcipient.

In particular, the invention relates to a pharmaceutical compositioncomprising a dye of formula (I), or a salt thereof, and one or morepharmaceutically acceptable adjuvants, excipients or diluents.

Alternatively, the invention relates to a pharmaceutical compositioncomprising a conjugate of formula (II) wherein R4 and/or R5 is C₁-C₆alkyl substituted with CONH—(S)_(m)-T as defined above, or a saltthereof, and one or more pharmaceutically acceptable adjuvants,excipients or diluents. Another aspect of this invention relates to adiagnostic kit comprising a compound of formula (I) or (II) as definedabove. In addition, the kit can contain additional adjuvants forimplementing the optical imaging. These adjuvants are, for example,suitable buffers, vessels, detection reagents or directions for use. Thekit preferably contains all materials for an intravenous administrationof the compounds of the invention.

The compounds of the invention may be administered either systemicallyor locally to the organ or tissue to be imaged, prior to the imagingprocedure. For instance, the compounds can be administeredintravenously. In another embodiment they may be administeredparenterally or enterally.

The compositions are administered in doses effective to achieve thedesired optical image of a tumor, tissue or organ, which can varywidely, depending on the compound used, the tissue subjected to theimaging procedure, the imaging equipment being used and the like.

The exact concentration of the imaging agents is dependent upon theexperimental conditions and the desired results, but typically may rangebetween 0.000001 mM to 0.1 mM. The optimal concentration is determinedby systematic variation until satisfactory results with minimalbackground fluorescence are obtained.

Once administered, the imaging agents of the invention are exposed to alight, or other form of energy, which can pass through a tissue layer.Preferably the radiation wavelength or waveband matches the excitationwavelength or waveband of the photosensitizing agent and has lowabsorption by the non-target cells and the rest of the subject,including blood proteins. Typically, the optical signal is detectableeither by observation or instrumentally and its response is related tothe fluorescence or light intensity, distribution and lifetime.

Description of the Syntheses

The preparation of the compounds of formula (I) or (II), as such or inthe form of physiologically acceptable salts, represents a furtherobject of the invention. The cyanine dyes and dye-conjugates of theinvention can be prepared for instance according to the methodsdescribed in the following sections and in the experimental part.

A general teaching about the preparation of cyanine dyes can be found inMujumdar R. B. et al., Bioconjugate Chem. 1993, 4(2): 105-111, whichrelates to the synthesis and labeling of sulfoindocyanine dyes. However,the cyanines of the present invention are characterized by a specificfunctionalization pattern not present in the compounds of the art, forwhich the set up of a proper synthetic approach was required. In fact,unlikely other known cyanines, the compounds of the invention bears eventhree functional moieties (carboxylic acid or amido groups) to bederivatized in different ways, so that the use of protecting groups isnecessary in most cases to direct the reactions on the desiredfunctional group.

It is known that difficulties can arise when manipulating the cyaninesat the strong pH and temperature conditions necessary for the removal ofthe protecting groups, since the stability of thecyclohexenyl-polymethine scaffold can be compromised in some cases, withsevere degradation of the dyes.

Moreover, further obstacles can be encountered due to a possiblehydrolysis and degradation of the amide groups —CONH—Y when deprotectinga carboxylic group of R1-R5 (typically, amide derivatives can hydrolyzein concentrated alkaline medium, see for instance Yamana et al, Chem.Pharm. Bull., 1972, 20(5), 881-891).

In one preferred embodiment, the protective group for the moiety R4 orR5 is an ester group. More preferably, an ethyl ester group can beadvantageously used.

Preparation of cyanine dyes of formula (I)

According to the invention, compounds of formula (I) can be preparedthrough the general sequence of synthetic steps as reported in thefollowing Scheme 1.

In the above Scheme 1, R1, R2, R3, X and Y are as defined above and Pgis absent or is a suitable protecting group.

Accordingly, a process of the present invention comprises the followingsteps:

-   -   a) treating suitable amounts of the        5-carboxy-2,3,3-trimethylindolenine of formula (III) and (IV)        with a polyhydroxylated amine, such as for instance glucamine,        meglumine, glucosamine, trometamol, serinol or isoserinol,        bearing suitable protecting groups on the hydroxy moieties; b)        reacting the intermediate (V) and the intermediate (VI) obtained        in steps a), together with        2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene, to obtain        the cyanine intermediate of formula (VII), wherein R1, R2, Y and        Pg are as defined;    -   c) optionally removing the protecting groups (Pg) of the Y        groups from the intermediate (VII);    -   d) substituting the chloro atom on the intermediate (VII) with a        suitable nucleophile to obtain the final product of formula (I)        or a salt thereof.

According to step(s) a) the reaction of derivatives (III) and (IV) withthe polyhydroxylated amine can be carried out by activation of thecarboxylate group with a coupling agent, for instance selected fromHATU, TBTU, HBTU, PyBOP, DCC, DSC and DCC-NHS, and an organic base, suchas TEA, DIPEA, NMM or pyridine, in a solvent such as dimethylformamide,dimethylacetamide, dimethylsulfoxide, acetonitrile etc, at roomtemperature for a suitable time ranging from 30 minutes to severalhours. This derivatization of the carboxylic acid can be performed onthe alkylated indolenine or on the indole, prior quaternarization. Inthis case, it is important to protect the hydroxyl groups of thepolyhydroxilated amines with a suitable protecting group such as acetyl,before the alkylation with sultone or bromo-hexanoic acid. Thisalkylation can be performed neat or in a high boiling solvent, such asbutyrronitrile, sulfolane, 1,2-dichlorobenzene, dimethylacetamide,dimethylformamide or dimethylsulfoxide, stirring the solution at hightemperature, for instance between 90° C. and 180° C., for several hours,typically from 12 hours to 5 days.

According to step b) the reaction can be performed using the Vilsmeierreagent in the bis anilido form or in the bis aldehyde form (as reportedin Scheme 1). The reaction can be carried out in several solvents suchas for example ethanol, methanol, acetic anhydride or acetic acid, withor without the addition of different bases, such as trimethylamine,pyridine, sodium acetate, potassium acetate etc., stirring the mixtureat different temperatures ranging from 45° C. to 120° C. for severalhours (typically 2-24 hours).

According to step c), any protecting group of intermediate (VII) isremoved from the moieties Y according to the known procedures, describedfor instance in T. W. Green and P. G. M. Wuts, Protective Groups inOrganic Synthesis, Wiley, N.Y. 2007, 4^(th) Ed., Ch. 5. Differently fromother classes of cyanines, these dyes showed higher stability at acidicpHs even at higher temperatures and several hours.

According to step d) the reaction can be performed using severalprotocols, depending on the X-R3 substituent. When phenol or itsderivative such as phenol-SO₃H is introduced, the dye can be heated inDMSO in the presence of an inorganic base, such as sodium or potassiumcarbonate. Whereas, when the chloro is replaced by phenyl or itsderivative, the reaction can be run in degassed water or a mixture ofdegassed water and a co-solvent, such as methanol, ethanol, etc.,heating for shorter time in the presence of a Pd catalyst, such as Pdacetate or Pd tetrakys and optionally a base, such as sodium orpotassium carbonate.

When R1 has the same meaning of R2, only one reaction a) is carried outand the subsequent step b) is performed with two units of intermediate(V) or (VI) instead of one unit of intermediate (V) and one unit ofintermediate (VI).

Alternatively, when R1 has the same meaning of R2 and is a linear orbranched C₁-C₆ alkyl substituted by —SO₃H, the compounds of formula (I)can be also prepared according to the following Scheme 2:

In the above Scheme 2, R1 is linear or branched C₁-C₆ alkyl substitutedby —SO₃H and X, Y and R3 are as defined above.

Accordingly, another process of the present invention comprises thefollowing steps:

-   -   f) reacting at least two equivalent the indolenine intermediate        (III), wherein R1 is linear or branched C₁-C₆ alkyl substituted        by —SO₃H, with the Vilsmeier reagent (in the bis aldehydic or        bis-anilido form) to obtain the corresponding cyanine        intermediate of formula (IX);    -   g) substituting the chloro atom on the intermediate (IX) with a        suitable nucleophile to obtain the intermediate (X);    -   h) treating suitable amounts of intermediate of formula (X) with        a polyhydroxylated amine, such as for instance glucamine,        meglumine, glucosamine, trometamol, serinol or isoserinol to        obtain the final product of formula (I) or a salt thereof.

According to step f) the reaction can be carried out in several solventssuch as for example ethanol, methanol, acetic anhydride or acetic acid,with or without the addition of different bases, such as trimethylamine,pyridine, sodium acetate, potassium acetate etc., stirring the mixtureat different temperatures ranging from 45° C. to 120° C. for severalhours (typically 2-24 hours). According to step g) the reaction can beperformed using several protocols, depending on the X-R3 substituent.When phenol or its derivative such as phenol-SO₃H is introduced, the dyecan be heated in DMSO in the presence of an inorganic base, such assodium or potassium carbonate.

Whereas, when the chloro is replaced by phenyl or its derivative, thereaction can be run in degassed water or a mixture of degassed water anda co-solvent, such as methanol, ethanol, etc., heating for shorter timein the presence of a Pd catalyst, such as Pd acetate or Pd tetrakys andoptionally a base, such as sodium or potassium carbonate.

According to step h) the reaction of derivative (X) with thepolyhydroxylated amine can be carried out by activation of thecarboxylate group with a coupling agent, for instance selected fromHATU, TBTU, HBTU, PyBOP, DCC, DSC and DCC-NHS, and an organic base, suchas TEA, DIPEA, NMM or pyridine, in a solvent such as dimethylformamide,dimethylacetamide, dimethylsulfoxide, acetonitrile etc, at roomtemperature for a suitable time ranging from 30 minutes to severalhours. In a further embodiment, a compound of formula (I), preparedaccording to the processes of the invention, can be convenientlyconverted into another compound of formula (I) by operating according towell-known synthetic conditions, the following being examples ofpossible conversions:

-   -   e) converting a compound of formula (I) wherein R2 is —COOH,        i.e. a compound of formula (Ib), into a corresponding compound        of formula (I) wherein R2 is —CONH₂, i.e. a compound of formula        (Ic):

According to step e), the conversion of a carboxylic acid of formula(Ib) into the corresponding carboxamide of formula (Ic) can beaccomplished in a variety of ways and experimental conditions, which arewidely known in the art for preparation of carboxamides. As an example,the carboxylic acid can be first converted in a suitable activated esterand then reacted with an ammonium salt, such as NH₄Cl, preferably in thepresence of a coupling agent, such as HBTU.

Preparation of conjugate compounds of formula (II)

The cyanine derivatives of formula (I), or salts thereof, can beconjugated with a suitable targeting moiety, optionally with theinsertion of a spacer, to obtain the corresponding compounds of formula(II). The conjugation can be accomplished following different proceduresknown in the art, such as for instance via direct coupling of acarboxylic acid group of the compounds with a nucleophilic residue ofthe targeting moiety, or optionally with the spacer, or by previousactivation, wherein the carboxylic acid group is transformed in a morereactive group, e.g. an ester such as NHS, before the coupling.

In one embodiment, in case of activation of a carboxylic acid throughformation of a NHS ester, the invention provides for a method oflabelling a targeting moiety using a dye of formula (XIa)

or a dye of formula (XIb)

wherein

-   -   X is direct bond or —O—;    -   Y is a group selected from linear or branched C₁-C₆ alkyl, C₃-C₇        cycloalkyl and heterocyclyl, substituted by at least two        hydroxyl groups;    -   R1 is a linear or branched C₁-C₆ alkyl substituted by a group        selected from —SO₃H, —COOH, —CONH₂ and —COO—C₁-C₆ alkyl; and    -   R3 is hydrogen, —SO₃H or a linear or branched C₁-C₆ alkyl        substituted by —COOH or —CONH—Y, wherein Y is a group selected        from linear or branched C₁-C₆ alkyl, C₃-C₇ cycloalkyl and        heterocyclyl, substituted by at least two hydroxyl groups,

the method comprising reacting the dye of formula (XIa) or (XIb) withthe targeting moiety.

If a compound of the formula (I) or (II) prepared according to theprocesses described above is obtained as mixture of isomers, theirseparation using conventional techniques into the single correspondingisomer of the formula (I) or (II) is within the scope of the presentinvention. The final compounds may be isolated and purified usingconventional procedures, for example chromatography and/orcrystallization and salt formation.

A compound of formula (I) or (II) as defined above can be converted intoa pharmaceutically acceptable salt. The compounds of formula (I) or (II)as defined above, or the pharmaceutically acceptable salt thereof, canbe subsequently formulated with a pharmaceutically acceptable carrier ordiluent to provide a pharmaceutical composition.

Experimental Part

The invention and its particular embodiments described in the followingpart are only exemplary and not to be regarded as a limitation of thepresent invention: they show how the present invention can be carriedout and are meant to be illustrative without limiting the scope of theinvention.

Materials and Equipment

All chemicals and solvents used for the reactions were reagent grade.Analytical grade solvents were used for chromatographic purifications.Most of the reagents, unless reported otherwise, are commercialproducts, including the targeting moieties (e.g. Panitumumab (Vectibix,Amgen; CASNr: 339177-26-3); c(RGDfK) (Cyclo(Arg-Gly-Asp-D-Phe-Lys),Bachem; CAS Nr: 161552-03-0). All synthesized compounds were purified byreverse phase chromatography (RP-HPLC) and characterized by massspectroscopy using a LC/MS instrument equipped with a UV-VIS detectorand an ESI source. Analysis were performed with a Waters Atlantis dC18 5μm, 4.6×150 mm column using a gradient of phase A CH₃COONH₄ 10 mM andphase B acetonitrile. Measured mass/charge ratios are listed for eachcompound.

A dual-beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer) was usedto determine the absorbance (Abs) of the compounds of the invention.Emission/excitation (Em/Ex) spectra and absolute fluorescence quantumyield (ϕ) measurements were carried out on a spectrofluorometer(FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018integrating sphere accessory. The measurements were performed using anexcitation wavelength at maximum absorbance of different dyes, and thesample was excited with a 450 W Xenon Light Source. Detection wasperformed by photomultiplier tubes (PMT-NIR) cooled detector or byTBX-04 detector. Dye solutions were carefully prepared to have anabsorbance lower than 0.1 (optical densities) to minimize re-absorptionphenomena.

In vivo imaging experiments were performed using the IVIS Spectrum InVivo Imaging System (Perkin Elmer Inc.). The system is equipped withwith 10 narrow band excitation filters (30 nm bandwidth) and 18 narrowband emission filters (20 nm bandwidth) spanning 430-850 nm.

List of abbreviations DCC N,N′-dicyclohexylcarbodiimide DIPEAN,N-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl sulfoxideDSC N,N′-Disuccinimidyl carbonate EuK Glutamic acid-urea-lysine HATU1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate HBTUO-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphateHPLC High performance liquid chromatography PBS Phosphate bufferedsaline NHS N-hydroxysuccinimide NMM N-methylmorpholine RT Roomtemperature PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate TEA Triethylamine TBTU2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborateTSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate μLMicroliter μM Micromolar t_(R) Retention time (HPLC) cRGDfKCyclo-(Arg-Gly-Asp-D-Phe-Lys)

The abbreviations for individual amino acids residues are conventional:for example, Asp or D is aspartic acid, Gly or G is glycine, Arg or R isarginine. The amino acids herein referred to should be understood to beof the L-isomer configuration unless otherwise noted (for instance Pheor “f” of the c(RGDfK) is in the form of D-isomer, as indicated in thelist of abbreviations).

Example 1: Synthesis of Compound 11 Preparation of intermediate (Va)

5-carboxy-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (10.8 g, 31.9mmol) was suspended in dry DMF (100 mL) under N₂ atmosphere: D-Glucamine(6.9 g, 38.2 mmol), DIPEA (11 mL, 63.7 mmol) and HATU (14.5 g, 38.2mmol) were added. The solution was stirred at RT for 16 hours, then colddiethyl ether (200 mL) was added. The mixture was filtered and the solidwas washed with ethyl acetate (2×50 mL). The solid was dissolved inwater and purified by flash chromatography on pre-packed C18 silicacolumn with 0.1% ammonium acetate/acetonitrile gradient. Fractionscontaining pure product were combined, distilled under vacuum andfreeze-dried three times, giving a pale pink solid (13.6 g, 80% yield)HPLC purity at 270 nm: 94%. MS: [M+H]⁺ 504.2.

Preparation of intermediate (VIIa)

Acetic acid (8.65 mL) was added to a suspension of intermediate (Va)(2.0 g, 3.98 mmol) in acetic anhydride (10 mL). The mixture was heatedat 45° C. and 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene(377.8 mg, 2.19 mmol) was added obtaining a yellow solution. Thetemperature was increased up to 75° C. (the solution became green-brown)and sodium acetate (408 mg, 4.97 mmol) was added obtaining immediately agreen solution. The solution was stirred 3 hours at 100° C., then it wascooled down to RT and the solvents were removed under reduced pressure.The crude was dissolved in water-acetonitrile and purified by flashchromatography on pre-packed C18 silica column with a water-acetonitrilegradient. Fractions containing the pure product were combined anddistilled under vacuum, giving a green solid (2.5 g, 40% yield). HPLCpurity at 780 nm: 90%. MS: [M+H]⁺ 1560.5.

Preparation of intermediate (VIIIa)

Intermediate (Vila) (2.5 g, 1.59 mmol) was dissolved inwater/acetonitrile 1/1 (20 mL) and the pH was adjusted from 2.2 to 1.5with 1N HCl. The solution was stirred at 80° C. for 16 hours. The crudewas purified by flash chromatography on pre-packed C18 silica columnwith a water-acetonitrile gradient. Fractions containing the pureproduct were combined, distilled under vacuum and freeze-dried,obtaining a green solid (1.09 g, 60% yield). HPLC purity at 780 nm 95%.MS: [M+H]+ 1140.7.

Synthesis of compound 11

To a solution of Intermediate (VIIIa) (1.09 g, 0.95 mmol) in degassedwater (20 mL), 4(2-carboxyethyl)-benzenboronic acid (332 mg, 1.71 mmol),Pd(PPh₃)₄ (165 mg, 0.14 mmol) and sodium carbonate (181 mg, 1.71 mmol)were added. The mixture was stirred at 80° C. under nitrogen atmospherefor 16 hours. Then, after cooling at RT, pH was adjusted to 6.5 with 2NHCl. The crude mixture was purified by flash chromatography onpre-packed C18 silica column with a water-acetonitrile gradient.Fractions containing the pure product were combined, distilled undervacuum and freeze-dried, obtaining a green solid (0.714 g, 60% yield).HPLC purity at 780 nm: 99%. MS: [M+H]+ 1253.5.

Example 2: Synthesis of Compound 12

Compound 11 as prepared in example 1 (56 mg, 0.0434 mmol) was suspendedin dry DMSO (5 mL) under nitrogen atmosphere. NMM (19 μL, 0.174 mmol),HATU (66 mg, 0.174 mmol) and D-glucamine (39 mg, 0.217 mmol) were added.After stirring 2 hours at RT, the mixture reaction was precipitated incold ethyl acetate (30 mL), the green solid was dissolved in water andpurified by flash chromatography on pre-packed C18 silica column with a0.1% ammonium acetate-acetonitrile gradient. Fractions containing theproduct were combined, distilled under vacuum and freeze-dried threetimes, giving a green solid as ammonium salt (69.88 mg). In order toremove the ammonium counterions, the solid was dissolved in water,charged on a C18 cartridge and washed with water (2 CV), 0.1% HCOOH (2CV), water (5 CV) and eluted with water/acetonitrile 1/1. Solvents weredistilled under vacuum and freeze-dried, obtaining a green solid (37 mg,60% yield). HPLC purity at 780 nm: 100%. MS: [M+H]⁺ 1419.4.

Example 3: Synthesis of Compound 13 Preparation of intermediate (VIb)

In a dried round bottom flask, 2,3,3-trimethyl-3H-indole-5-carboxylicacid (711 mg, 3.5 mmol) was solubilized in dry DMF (4 mL) under nitrogenatmosphere, then DIPEA (380 μL, 4.90 mmol) was added. After 30 minutesof stirring at RT, a solution of TBTU (603 mg, 4.20 mmol) in dry DMF (2mL) was added. After 1 hour of stirring at RT, a suspension ofD-glucamine (312 mg, 3.85 mmol) in dry DMF (2 mL) was added. After onenight the reaction was not complete, therefore the same amount of TBTU,DIPEA and D-glucamine were added and stirred for other 2 hours. Themixture was dried under vacuum and purified on pre-packed C18 silicacolumn with a water-acetonitrile gradient. Fractions containing the pureproduct were combined, distilled under vacuum and freeze-dried,obtaining a white-brown powder (888.2 mg, 70% yield). HPLC purity at 270nm 93% purity, MS: [M+H]⁺ 502.57.

Acetic anhydride (3 mL) and pyridine (0.5 mL) were added to thisintermediate (888.2 mg, 2.42 mmol), resulting in a suspension thatgradually solubilized during time. The mixture was kept under stirringin a nitrogen atmosphere at RT for 4 hours. The solution wasconcentrated under vacuum and purified on a pre-packed C18 silica columnwith a water-acetonitrile gradient. Fractions containing the pureproduct were collected, concentrated under vacuum and freeze-dried,obtaining a white-brown solid (861.4 mg, 62% yield). HPLC purity at 270nm 97.5%, MS: [M+H]⁺ 204.

In a round bottom flask, such obtained product (1.103 g, 1.91 mmol) and1-bromohexanoic acid (933 mg, 4.77 mmol) were solubilized in1,2-dichlorobenzene (6 mL). The mixture was heated at 130° C. undernitrogen atmosphere for 6 hours, then other 1-Bromohexanoic acid (933mg, 4.77 mmol) was added and the reaction was kept at the sameconditions overnight. The crude was washed with diethyl ether, thesolvent was decanted, the solid was dissolved in acetonitrile andpurified on pre-packed C18 silica column with a water-acetonitrilegradient. Fractions containing the product were collected andconcentrated in vacuum, to give a red oil (861.4 mg, 62% yield). HPLCpurity at 270 nm 97.5%, MS: [M+H]⁺ 691.0.

Preparation of Intermediate (VIIb)

In a dried round bottom flask, intermediates (Va) (1.170 g, 2.33 mmol),prepared as in example 1, and (VIb) (1.79 g, 2.33 mmol) were suspendedin acetic anhydride (20 mL) and acetic acid (5 mL). The mixture washeated at 45° C., until the two powders were fully solubilized. Then,2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (430 mg,2.49 mmol) was added and the mixture was heated up to 50° C. Potassiumacetate (237 mg, 2.89 mmol) was added and the mixture was heated at 100°C. for 2 hours. The solvents were removed under vacuum and the crudegreen solid was purified on a pre-packed C18 silica column with awater-acetonitrile gradient. Fractions containing the product werecollected, concentrated in vacuum and freeze-dried, to give a greenpowder (1.221 g, 35% yield). HPLC purity at 780 nm 72%, MS: [M+H]⁺1540.6.

Preparation of intermediate (VIIIb)

Intermediate (VIIb) (701 mg, 0.33 mmol) was solubilized in acetonitrile(3 mL) and water (15 mL) was added. The solution was acidified at pH 1.6with 1N HCl and was heated at 80° C. for 4 hours, then at 55° C.overnight. The organic solvent was removed under reduced pressure andthe aqueous solution was purified on a pre-packed C18 silica column witha water-acetonitrile gradient. Fractions containing the product werecollected, concentrated under vacuum and freeze-dried to give a greenpowder (179 mg, 50% yield). HPLC purity at 780 nm 95%, MS: [M+H]⁺1120.3.

Synthesis of Compound 13

In a dried round bottom flask, intermediate (VIIIb) (115 mg, 1.03 mmol)was solubilized in degassed water (3 mL), then phenylboronic acid (22.7mg, 1.85 mmol), sodium carbonate (19.6 mg, 1.85 mmol) and palladiumtetrakis (17.8 mg, 0.15 mmol) were added. The mixture was heated at 80°C. under nitrogen atmosphere for 2 hours. The solution was brought at pH7.15 with 0.1 N HCl and purified on a pre-packed C18 silica column witha water-acetonitrile gradient. Fractions containing the product werecollected, concentrated under vacuum and freeze-dried, giving a greenpowder (99 mg, 83% yield). HPLC purity at 780 nm 98%, MS: [M+H]⁺ 1162.2.

Example 4: Synthesis of Compound 14

A solution of intermediate (VIIIb) prepared as in example 2 (120 mg,0.107 mmol) in dry DMSO (5 mL) was dropped into a suspension of phenol(101 mg, 1.07 mmol) and anhydrous potassium carbonate (148 mg, 1.07mmol) in dry DMSO (7 mL) under nitrogen atmosphere. The mixture wasstirred at 50° C. for 4 hours. After cooling to RT, cold diethyl ether(30 mL) was added, the solid was filtered and washed twice with colddiethyl ether. It was dissolved in water and the pH was adjusted from11.5 to 6 with 0.5N HCl. The crude solid was purified by flashchromatography on pre-packed C18 silica column with a 0.1% ammoniumacetate-acetonitrile gradient. Fractions containing the pure productwere combined and distilled under vacuum. In order to remove theammonium counterions pH was adjusted to 1.6, the product was charge on aC18 silica cartridge, washed with water and eluted withwater-acetonitrile 1:1. Solvents were distilled under vacuum and theaqueous solution was freeze-dried giving a green solid (42 mg, 33%yield). HPLC purity at 780 nm 99.3%. MS: [M+H]⁺ 1178.3.

Example 5: Synthesis of Compound 15

A solution of intermediate (VIIIb) prepared as in example 2 (20 mg,0.018 mmol) in dry DMSO (3 mL) was dropped into a suspension of4-hydroxybenzene sodium sulfonate (35 mg, 0.18 mmol) and anhydrouspotassium carbonate (25 mg, 0.18 mmol) in dry DMSO (5 mL) under nitrogenatmosphere. The mixture was stirred at 50° C. for 4 days. Cold diethylether (30 mL) was added to the brown mixture, the solid was filtered andwashed twice with cold diethyl ether. The solid was dissolved in waterand pH was adjusted from 11.5 to 6 with 0.5N HCl: the solution turnedgreen again. The crude was purified by flash chromatography onpre-packed C18 silica column with a water-acetonitrile gradient.Fractions containing the pure product were combined, distilled undervacuum and freeze-dried giving a green solid (15 mg, 65% yield). HPLCpurity at 780 nm 100%. MS: [M+H]⁺ 1257.3.

Example 6: Synthesis of Compound 16

A solution of intermediate (VIIIa) prepared as in example 1 (30 mg,0.026 mmol) in dry DMSO (3 mL) was dropped into a suspension of4-hydroxybenzene sodium sulfonate (14 mg, 0.08 mmol) and anhydrouspotassium carbonate (10 mg, 0.08 mmol) in dry DMSO (3 mL) under nitrogenatmosphere. The mixture was stirred at 80° C. for 6 hours. Cold ethylacetate (20 mL) was added to the brown mixture, the solid was filtered,dissolved in water and pH was adjusted from 11.5 to 3 with 0.5N HCl: thesolution turned green again. The crude was purified by flashchromatography on pre-packed C18 silica column with a water-acetonitrilegradient. Fractions containing the pure product were combined, distilledunder vacuum and freeze-dried giving a green solid (14 mg, 42% yield).HPLC purity at 780 nm 97.8%. MS: [M+H]⁺ 1277.3.

Example 7: Synthesis of Compound 17

In a dried round bottom flask, intermediate (VIIIa) prepared as inexample 1 (9 mg, 0.008 mmol) was solubilized in degassed water (2 mL),then phenylboronic acid (2.2 mg, 0.018 mmol) and palladium acetate (0.15mg, 0.0006 mmol) were added. The mixture was refluxed under nitrogenatmosphere for 2 hours. The solution was purified on a pre-packed C18silica column with a water-acetonitrile gradient. Fractions containingthe product were collected, concentrated under vacuum and freeze-dried,giving a green powder (7 mg, 75% yield). HPLC purity at 780 nm 98%, MS:[M+H]⁺ 1184.3.

Example 8: Synthesis of Compound 19 Preparation of Intermediate (IXa)

Compound (111a) (180 mg, 0.53 mmol),N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]anilinemonohydrochloride (101 mg, 0.265 mmol) and sodium acetate (109 mg, 1.32mmol) were dissolved in absolute ethanol (55 mL) and the solution wasrefluxed for 26 hours. The solvent was removed under reduced pressureand the crude was purified by flash chromatography on silica C18 columnusing a gradient of water/acetonitrile. Fractions containing the pureproduct were combined, distilled under vacuum and freeze-dried threetimes, obtaining a green solid (154 mg, 72% yield). HPLC purity at 780nm 97%. MS: [M+H]⁺ 815.2.

Preparation of Intermediate (Xa)

A solution of intermediate (IXa) (40 mg, 0.05 mmol) in dry DMSO (4 mL)was dropped into a suspension of phenol (45 mg, 0.49 mmol) and anhydrouspotassium carbonate (68 mg, 0.49 mmol) in dry DMSO (8 mL) under nitrogenatmosphere. The mixture was stirred at 50° C. for 8 hours. After coolingto RT, cold diethyl ether (30 mL) was added, the solid was filtered andwashed twice with cold diethyl ether. It was dissolved in water and thepH was adjusted from 12 to 6 with 0.5N HCl. The crude solid was purifiedby flash chromatography on pre-packed C18 silica column with awater/acetonitrile gradient. Fractions containing the pure product werecombined and distilled under vacuum. Solvents were distilled underreduced pressure and the aqueous solution was freeze-dried giving agreen solid (26 mg, 61% yield). HPLC purity at 780 nm 98%. MS:[M+H]⁺872.1

Synthesis of Compound 19

TBTU (5.1 mg, 0.015 mmol) was added to a solution of intermediate (Xa)(3.3 mg, 0.0038 mmol) and DIPEA (6 μL, 0.031 mmol) in anhydrous DMF (5mL). The solution was stirred at RT for 30 minutes, then serinol (1 mg,0.011 mmol) was added and the solution was stirred for 90 minutes. Thesolvent was removed under reduced pressure and the crude was purified byflash chromatography on silica C18 column eluting with a gradient ofwater-acetonitrile. Fractions containing the pure product were combined,concentrated under reduced pressure and freeze-dried, obtaining a darggreen solid (2 mg, 53% yield). HPLC purity at 780 nm 98.5%. MS:[M+H]⁺1019.1.

Analogously, the compounds 18, 20, 21, 22 and 23 were prepared byfollowing a similar procedure, but using a different hydroxylated amine(as Y groups) or starting from a scaffold bearing a phenyl instead ofphenol, as described above.

Example 9: Synthesis of Compound 1

Compound 11 (5 mg, 0.004 mmol) was dissolved in dry DMF (3 mL) underinert atmosphere. DIPEA (6.8 μL, 0.04 mmol) and TSTU (12 mg, 0.04 mmol)were added and the solution was stirred for 48 hours at RT. Then, colddiethyl ether (20 mL) was added and the precipitate was filtered andwashed twice with cold diethyl ether, obtaining a green solid. HPLCpurity at 780 nm: 90%. MS: [M+H]⁺ 1351.

The NHS ester was dissolved in a solution of c(RGDfK) (2.7 mg, 0.004mmol) in 1 mL of borate buffer at pH 9. The reaction was stirred for 24hours at RT, then the product was precipitate in cold diethyl ether andwashed twice with cold diethyl ether, obtaining a green solid. The crudesolid was purified by HPLC chromatography on Kromasil C8 column using alinear gradient of 0.1% ammonium acetate/acetonitrile. Fractionscontaining the pure product were combined, distilled under vacuum andfreeze-dried three times, obtaining a green solid (3.9 mg, 50% yield).HPLC purity at 780 nm: 97.6%. MS: [M+H]⁺ 1840.6.

Example 10: Synthesis of Compound 3 Procedure a Via Direct Coupling

In a dry round bottom flask, compound 13 (2 mg, 0.0017 mmol) wassolubilized in DMF (1 mL) with DIPEA (0.6 μl, 0.003 mmol) at RT undernitrogen flow. After 30 minutes of stirring at RT, a solution of TBTU(0.6 mg, 0.002 mmol) in dry DMF (1 mL) was added. After 1 hour ofstirring at RT, a solution of c(RGDfK) (1.4 mg, 0.002 mmol) in dry DMF(1 mL) was dropped into the stirring solution. The reaction was stirredovernight at RT, then the solvent was removed under vacuum and the crudewas purified on analytical HPLC C18 silica column with 0.1% ammoniumacetate-acetonitrile gradient. Fractions containing the pure productwere combined, concentrated under vacuum and freeze dried three times,obtaining a green powder (0.7 mg, 23% yield). HPLC purity at 780 nm 98%,MS: [M/2]⁺872.8.

Procedure B Via NHS Ester

In a dry round bottom flask, compound 13 (5 mg, 0.0043 mmol) wassolubilized in dry DMF (2 mL) with DIPEA (2.2 μl, 0.0129 mmol) and asolution of TSTU (3.78 mg, 0.0129 mmol) in dry DMF (1 mL) was added atroom temperature under nitrogen flow. The green solution was stirredovernight at RT, then the product was precipitated by addition of colddiethyl ether. The solid was centrifuged, washed twice with diethylether and used in the following step with no further purification. TheNHS ester was dissolved in a solution of c(RGDfK) (2.2 mg, 0.0043 mmol)in borate buffer at pH 9 (1 mL) and the solution was stirred at roomtemperature overnight. Then, pH was adjusted at 7 with 0.1 N HCl and thecrude was purified on analytical HPLC C18 silica column with 0.1%ammonium acetate-acetonitrile gradient. Fractions containing the pureproduct were combined, concentrated under vacuum and freeze dried threetimes, obtaining a green powder (3.9 mg, 52% yield). HPLC purity at 780nm 97.7%, MS: [M/2]⁺872.8.

Example 11: Synthesis of Compound 2

Compound 15 (6 mg, 0.0048 mmol) was suspended in dry DMF (6 mL), thenNMM (2.6 μL, 0.024 mmol) and TSTU (7.2 mg, 0.024 mmol) were added. Thesolution was stirred at RT for 16 hours. Cold diethyl ether (30 mL) wasadded, the precipitate was filtered and washed twice with cold diethylether. The green solid was used in the following step without anyfurther purification, HPLC at 780 nm: 88%, MS: [M+H]⁺ 1354.3.

The NHS ester was dissolved in borate buffer at pH 9 (1 mL) and asolution of c(RGDfK) (3.4 mg, 0.0048 mmol) in borate buffer at pH 9 (1mL) was added. The solution was stirred at RT for 5 hours, then pH wasadjusted to 6.5 with 0.1 N HCl and the crude was purified by HPLCchromatography on Phenyl-C18 column using a linear gradient of 0.1%ammonium acetate-acetonitrile. Fractions containing the pure productwere combined, distilled under vacuum and freeze-dried three times,obtaining a green solid (6.4 mg, 71% yield). HPLC purity at 780 nm99.6%. MS: [M+H]⁺ 1845.5.

Example 12: Synthesis of Compound 4

Compound 14 (12 mg, 0.0102 mmol) was dissolved in dry DMF (1 mL) undernitrogen atmosphere. DIPEA (4.8 μL, 0.0286 mmol) and TBTU (4.6 mg,0.0143 mmol) were added and after 1 hour of stirring at RT c(RGDfK)(6.15 mg, 0.0102 mmol) was added. The reaction was stirred at RT for 16hours, then cold diethyl ether (25 mL) was added, the precipitate wasfiltered and washed twice with cold diethyl ether. The crude solid wasdissolved in water and purified by HPLC chromatography on Phenyl-C18column using a linear gradient of 0.1% ammonium acetate-acetonitrile.Fractions containing the pure product were combined, distilled undervacuum and freeze-dried three times, obtaining a green solid (12.3 mg,67% yield). HPLC purity at 780 nm 98.3%, MS: [M+H]⁺ 1763.6.

Example 13: Synthesis of Compound 5

The monoclonal antibody EGFR ligand Panitumumab (6 mg) was diluted up to5 mg/mL in PBS and pH was adjusted by adding 120 μL of 1.0 M potassiumphosphate pH 9. Compound 15-NHS ester (prepared as described forcompound 2 in Example 11) was dissolved in DMSO at a concentration of 10mg/ml; then the dye and the antibody were immediately mixed at a molarratio of 2.5:1 and kept at room temperature in the dark for 3 h. After 3h, the conjugation reaction mixture was layered onto phosphate bufferedsaline (PBS)-equilibrated Zeba Spin columns and centrifuged at 1500 gfor 2 min to separate the conjugate from the free dye. After filtrationthrough a 0.22-μm polyethersulfone (PES) membrane, the conjugatedPanitumumab solution in PBS at pH 7.4 was analyzed by SE-HPLC, RP-HPLC,UV/VIS spectrophotometry to determine concentration and purity. Themolar conjugation ratio (dyes molecules coupled per antibody) was 1.53.

Example 14: Synthesis of Compound 6

The small molecule CAIX ligand 4a, described in Wichert et al., Nat Chem2015, 7, 241-249, was prepared according to the procedure thereindisclosed and conjugated to compound 15. 11 mg of compound 15 (8.7 μmol)were dissolved in 1 mL of DMF, then 4.5 mg of PyBOP (8.7 μmol) and 6 μLof DIPEA (35.0 μmol) were added under continuous stirring. After 20minutes, 8 mg of small molecule 4a (13.0 μmol) were dissolved in 1 mL ofDMF and added to the reaction mixture which was stirred for additional30 minutes at room temperature. Purification was performed bypreparative HPLC with a yield of 50%. The isolated pure product wascharacterized by HPLC-UV-VIS-MS-ESI (+) using a Waters Atlantis dC18column (μm, 4.6×150 mm). HPLC purity at 779 nm: 99%; MS: [M/2]⁺929.7.

Example 15: Synthesis of Compound 7

The small molecule CAIX ligand 8a, described in Wichert et al., Nat Chem2015, 7, 241-249, was prepared according to the procedure thereindisclosed and conjugated to Compound 15. 9 mg of Compound 15 (7.1 μmol)were dissolved in 1 mL of DMF, then 3.7 mg of PyBOP (7.1 μmol) and 5 μLof DIPEA (28.0 μmol) were added under continuous stirring. After 20minutes, 11 mg of molecule 8a (11.0 μmol) were dissolved in 1 mL of DMFand added to the reaction mixture which was stirred for additional 30minutes at RT. The purification was performed by preparative HPLC with ayield of 50%. The isolated pure product was characterized byHPLC-UV-VIS-MS-ESI (+) using a Waters Atlantis dC18 column (μm, 4.6×150mm). HPLC purity at 780 nm: 99%; MS: [M/2]+ 1157.8.

Example 16: Synthesis of Compound 8

9.9 mg of Compound 15 (7.9 μmol) was dissolved in 3 mL of dry DMF, then2 μL of NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol) were added andthe mixture was stirred for 2 h at RT. The NHS ester of Compound 15(HPLC conversion 85.9%) was precipitated in 25 mL of ice cold ethylacetate. The precipitate was washed with ethyl acetate and dried underN₂ flow.

The NHS ester of Compound 15 was dissolved in 1 mL of dry DMF. Asolution prepared by dissolving 5.74 mg of EuK TFA salt (13.43 μmol) in1 mL of DMF was dropped. Then, a solution of 13.72 μL DIPEA (7.8 μmol)in 1 mL DMF was dropped. The solution was allowed to stir overnight atRT under N₂ atmosphere. The product (HPLC conversion 84%) wasprecipitated in 25 mL of ice cold diethyl ether and purified on apre-packed silica C18 column (BIOTAGE® SNAP ULTRA 26 g) with anautomated flash chromatographic system (Combiflash Rf+) eluting with awater/acetonitrile gradient. Fractions containing the desired pureproduct were combined, concentrated under vacuum and freeze-dried,recovering 6.65 mg of a green solid (HPLC purity area %: 98.7% at 785 nmand 100% at 254 nm; [M−H]⁺1558.7). The yield from Compound 15 was 54.0%.

Example 17: Synthesis of Compound 9

9.9 mg of Compound 15 (7.9 μmol) was dissolved in 3 mL of dry DMF. 2 μLof NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol) were added and themixture was stirred for 2 h at RT. The NHS ester of Compound 15 (HPLCconversion 85.9%) was precipitated in 25 mL of ice cold ethyl acetate.The precipitate was washed with ethyl acetate and dried under N₂ flow.

NHS ester of Compound 15 was dissolved in dry DMF (1 mL). A solution ofEuK-(3-(2-naphtyl)-alanine)-tranexamic acid TFA salt, prepared asdescribed in Benešová et al., J Nucl Med 2015, 56:914-920, (7.23 mg,0.00945 mmol) in DMF (1 mL) was dropped. Then, a solution of DIPEA (6.86μL, 0.039 mmol) in DMF (1 mL) was dropped. The solution was allowed tostir overnight at RT under N₂ atmosphere. The product (HPLC conversion93%) was precipitated in 25 mL of ice cold diethyl ether and purified ona pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 26 g) with anautomated flash chromatographic system (Combiflash Rf+) eluting with awater/acetonitrile gradient. Fractions containing the desired pureproduct were combined, concentrated under vacuum and freeze-driedrecovering 4.25 mg of a green solid (HPLC purity area %: 99.6% at 785 nmand 98% at 254 nm; [M−H]⁺1894.6). The yield from Compound 15 was 28.0%.

Example 18: Optical Properties

The compounds of the invention have been characterized in terms of theiroptical properties in vitro in aqueous medium (i.e., water/PBS pH 7.4)and in a clinical chemistry control serum (Seronorm, Sero SA), mimickingthe chemical composition and optical properties of human serum. All dyeor dye-conjugate solutions were freshly prepared. ICG and S0456 wereused as commercial references.

In particular, the excitation and emission maxima and the absolutefluorescence quantum yield (Φ) of representative compounds of formula(I) and conjugates of formula (II) are shown in Table II.

TABLE II Excitation/Emission maxima and absolute fluorescence quantumyields of compounds of formula (I) and (II) Max Ex/Em Φ Φ Dyes offormula (I) (PBS pH 7.4) (PBS pH 7.4) (Seronorm) ICG (Reference) 780/810nm 1.5%  7.8% S0456 (Reference) 782/803 nm 3.9%  7.7% Compound 11764/786 nm 6.3% 11.5% Compound 14 781/803 nm 4.4% 10.6% Compound 15781/804 nm 5.4%  8.2% Compound 17 768/788 nm 5.5% 11.8% Compound 19783/805 nm 6.5% 10.9% Compound 18 781/804 nm 6.2% 10.5% Compound 20780/801 nm 6.3%  7.7% Compound 21 769/790 nm 5.6% 12.8% Compound 22782/803 nm 6.1%  9.8% Conjugates of formula (II) Max Ex/Em Φ Φ (PBS pH7.4) (PBS pH 7.4) (Seronorm) Compound 1 771/788 nm 4.4% 10.9% Compound 2783/805 nm 6.1%  7.5% Compound 3 771/787 nm 6.4%  8.1% Compound 4781/803 nm 5.0%  9.9% n/a: not available

The compounds of the invention are characterized by absorption maximacomprised in the range from about 760 nm to 810 nm. The dyes are endowedwith fluorescence emission in the near-infrared region and highfluorescence quantum yield, even when conjugated to a targeting moiety.Overall, the dyes and conjugates display higher fluorescence quantumyield than ICG and S0456.

Example 19: Affinity to Human Albumin (HSA)

An analysis of the binding affinity of the compounds of the invention tohuman albumin was carried out and the results compared with ICG andS0456 as a reference. Binding affinity to human serum albumin (HSA;Sigma Aldrich, A9511) was measured using two methods, according to thelevel of binding affinity of the compounds.

The first method, optimal for compounds which strongly interact withHSA, is based on the analysis of the absorbance spectrum peak shiftafter the incubation of the dye in solutions containing HSA. Briefly,the samples were incubated at a fixed concentration (1 μM) with HSAdilutions (1×10−6-4×10−4 M), in phosphate buffer for 5 min in thespectrophotometer at 25° C. before measurements.

The measure was performed at the maximum absorbance wavelength of theshifted peak.

The second method, optimal for compound with low affinity for HSA, isbased on measuring the variation of the absorbance of solutionscontaining the dye and various concentrations of HSA afterultrafiltration. Briefly, each compound was incubated at a fixedconcentration (2 μM) with HSA dilutions (1×10⁻⁶-4×10⁻⁴ M), in phosphatebuffer. The samples were centrifuged (10,000 g for 30 min at 25° C.) ina Microcon device (10 kDa MWCO, Amicon Ultra-0.5 Centrifugal Filter Unitwith Ultracel-10 membrane, Millipore) and the absorbance measurements ofthe filtrates were obtained with the spectrophotometer at the maximumabsorbance wavelength of the fluorophore. For both methods, the affinityconstant (K_(A), M⁻¹) was calculated by fitting the raw data with thefollowing formula:

$\frac{\Delta A}{b} = \frac{{\Delta\varepsilon} \cdot {K_{RL}\lbrack L\rbrack} \cdot R_{t}}{{K_{RL}\lbrack L\rbrack} + 1}$

-   -   wherein    -   ΔA/b=Absorbance measured (b=1 cm)    -   K_(RL)=K_(A) calculated by regression analysis (curve fitting)    -   Δε·Rt calculated by regression analysis (curve fitting)    -   [L]=Albumin concentration

In the first method, ΔA/b corresponds to the absorbance measured foreach sample, whereas in the second method ΔA/b is obtained subtractingthe absorbance of the control sample (dye without HSA) to the absorbanceof each other sample.

Both methods have demonstrated to provide comparable results, as shownby parallel experiments conducted on the commercial cyanine dye IRDye800CW carboxylate (LI-COR Inc., Lincoln, USA) using the first method(HSA K_(A)=215,000 M⁻¹) and the second method (HSA K_(A)=216,000 M⁻¹).

However, the measurement of the affinity constant is more precise whenthe suitable method is used as a function of the affinity level of thecompound.

The results of the binding affinity measured for representativecompounds of the invention with one of the two methods are reported inTable III and compared with the results obtained for the cyanine dyeIRDye 800CW carboxylate dye as reference compound.

TABLE III Binding affinity to human serum albumin (HSA) HSA affinity(K_(A), M⁻¹) ICG (Reference) 347,000 S0456 (Reference) 350,000 Compound11 6,500 Compound 14 23,800 Compound 15 90,600 Compound 17 12,262Compound 18 14,600 Compound 19 28,300 Compound 20 13,800 Compound 2132,400 Compound 22 8,360 Compound 2 26,200 Compound 4 10,000

As shown in Table III, both the dyes and dyes-conjugates of theinvention display a remarkably low binding affinity to human albumincompared to the known near-infrared dyes ICG and S0456, with affinityconstants of one or two orders of magnitude lower.

This advantageous feature is preserved in the dyes of the invention evenwhen conjugated with a targeting moiety (for instance Compounds 2 and4), as the conjugation of the dyes with a targeting moiety does notaffect the affinity to human albumin.

Example 20: Receptor Binding Affinity

The binding affinity of the conjugates of formula (II) to a specificreceptor was determined to assess whether the targeting efficacy of themolecular vector is preserved after the labeling with the dyes of theinvention.

As example of small molecules and peptide/peptidomimetic conjugates, thereceptor affinity of representative integrin-binding conjugates wasevaluated through calculation of their IC₅₀ (half maximal inhibitoryconcentration), using an enzyme-linked immunosorbent assay (ELISA), aspreviously reported (Kapp et al., Sci. Rep. 2017, 7, 39805).

Briefly, 96-well ELISA plates were coated overnight at 4° C. with theextracellular matrix (ECM) protein Vitronectin in carbonate buffer (15mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6). Each well was then washed withPBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mMKCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.01% Tween20, pH 7.4) and blocked for1 h at RT with TS-B-buffer (Tris-saline/BSA buffer; 20 mM Tris-HCl, 150mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, pH 7.5, 1% BSA). In themeantime, a dilution series of the compound and internal standard wasprepared in an extra plate. After washing the assay plate three timeswith PBS-T, 50 μL of the dilution series were transferred to each well.50 μL of a solution of human recombinant integrin α_(v)β₃ (R&D Systems,1 μg/mL) in TS-B-buffer was transferred to the wells and incubated for 1h. The plate was washed three times with PBS-T buffer, and then primaryantibody anti-α_(v)β₃ was added to the plate. After incubation andwashing three times with PBS-T, the secondary anti-IgGperoxidase-labeled antibody was added to the plate and incubated for 1h. After washing the plate three times with PBS-T, the plate wasdeveloped by quick addition of 3,3′,5,5′-tetrametylbenzidine (TMB) andincubated for 5 min in the dark. The reaction was stopped with 3 MH₂SO₄, and the absorbance was measured at 450 nm with a plate reader(Victor3, Perkin Elmer).

The IC₅₀ of the representative Compounds 1, 2 and 4 was tested induplicate, and the resulting inhibition curves were analyzed usingGraphPad Prism version 4.0 for Windows (GraphPad Software). Theinflection point defines the IC₅₀ value. All experiments were conductedusing c(RGDfK) as internal standard.

The tested molecular probes, coupled to c(RGDfK), showed comparableaffinity to the human α_(v)β₃ receptor, and similar affinity to theunconjugated reference peptidomimetic c(RGDfK), as reported in Table IV.

TABLE IV Binding affinity to the human α_(v)β₃ integrin receptor ofcompounds of formula (II) compared to the peptidomimetic c(RGDfK).Receptor affinity (IC₅₀, nM ± St. Dev.) c(RGDfK) 2.69 ± 0.70 Compound 12.73 ± 0.50 Compound 2 1.64 ± 0.41 Compound 4 1.84 ± 0.38

Example 21: Cell Uptake

The human melanoma cell line WM-266-4 (ATCC, CRL-1676) was used as invitro model to assess the cell uptake of representative integrin-bindingCompounds 1, 2 and 4, based on the high expression of the integrinreceptors, particularly α_(v)β₃, on the membrane of these cells (Capassoet al., PlosOne 2014).

Adherent cells at about 70% confluence were incubated with the Compounds1 or 3 (1 μM) for 2 h at 37° C. (5% CO₂) in presence of Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mMglutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. After twowashing steps with PBS, cells were detached using 0.1 mM EDTA in PBS,centrifuged and suspended in buffer (PBS, 0.5% BSA, 0.1% NaN₃) for flowcytometry experiments. Fluorescence Activated Cell Sorting (FACS) wasused to detect the fluorescence signal within the cells, as measure ofcell uptake. Samples were excited with an Argon laser and the emissiondetected using a 670 nm longpass filter. Values of fluorescenceintensity were obtained from the histogram statistic produced by theinstrument software.

To assess the specificity of receptor-mediated cell uptake, experimentswere performed by incubating the cells with the molecular probes inpresence of high concentration (100 μM) of the unlabeled molecularvector c(RGDfK) as competitor. The residual internalization wascalculated by considering the value of fluorescence intensity in absenceof the competitor as 100%.

Furthermore, to assess the effect of biological fluids on the celluptake, parallel experiments were performed incubating the cells with acompounds of the invention in presence of human serum from male ABplasma (Sigma Aldrich, H4522). The residual internalization wascalculated by considering the value of fluorescence intensity in absenceof the serum as 100%. Such uptake assessment also represents anindication of the percentage of compound which is sequestered by theplasma proteins when it diffuses through the vascular compartment beforereaching the tissue of interest and the particular targeted receptor. InTable V the cell uptake performance of representative Compounds 2 and 4of the invention is shown.

The present compounds displayed high cell uptake in presence of humanserum. Thus, for the present compounds it is observed that theinternalization in the cells is receptor-mediated and is only slightlyaffected by the binding to human serum proteins, in particular albumin(about 10-20% of residual uptake), confirming the medium-to-low bindingaffinity to human albumin of the present compounds (K_(A)=about 1-6×10³M⁻¹, as shown in example 10). Furthermore, the Compounds of theinvention were compared with the reference compounds ICG-RGD (Capozza etal., Photoacoustic 2018, 11, 36-45) and ICG-c(RGDfK), prepared with thesame method for ICG-RGD. These results show that the compounds of thepresent invention have been surprisingly found endowed with a higherefficacy in cell internalization with respect to similar compounds knownin the art once incubated in presence of human serum.

TABLE V Uptake of the integrin-binding fluorescent probes of theinvention into WM-266-4 human melanoma cells. Residual cell uptake inpresence of human serum ICG-RGD 12% ICG-c(RGDfK)  8% Compound 2 90%Compound 4 85% n/a: not available.

Notably, neither the interaction of the present compounds with thereceptor on the cell surface, nor the internalization of thereceptor-probe complex within the cell were impaired by the structure ofthe conjugated dyes, and particularly by the presence in position Y ofthe compounds of moieties strongly hydrophilic and with high sterichindrance. Thus, the presence of the hydrophilic moieties on theconjugated dyes provide highly efficient and specific receptor bindingand probe internalization even in presence of plasma proteins, whichwould sequester a conjugate lacking the hydrophilic moieties andnegatively affect the binding efficiency.

Example 22: Tumor Uptake in Animal Models

Human Glioblastoma

Tumor uptake experiments were carried out in an animal model of humanglioblastoma (subcutanous) overexpressing the integrin receptors,particularly α_(v)β₃. Briefly, human glioblastoma U87MG cells (ATCC,HTB-14) were cultured in Eagle's Minimum Essential Medium (EMEM)supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 100IU/mL penicillin, 100 μg/mL streptomycin. Male Balb/c nu/nu mice, 4-6weeks of age (Charles River Laboratories), underwent subcutaneousimplantation (right flank) of about 10 million cells suspended in 0.1 mLof EMEM. Mice were housed 4 per cage with food and water ad libitum.Animals were fed with VRF1 (P) sterile diet (Special Diets Services Ltd)up to the end of the acclimation period (5 days). Then, AIN-76a rodentdiet irradiated (Research Diets), a special diet that reducesauto-fluorescence, was used up to the end of the experiments. Tumorgrowth was monitored by longitudinal assessments using a caliper up tothe target size of 300-600 mm³ (3-4 weeks after cell implantation).Imaging experiments were performed using the preclinical optical systemIVIS Spectrum (Perkin Elmer). In vivo imaging was performed under gasanesthesia (Sevofluorane 6-8% in oxygen). Animals were intravenouslyinjected (lateral tail vein) with the compounds, and euthanized 24 hpost-administration. Regions of interest (ROIs) were drawn on theexcised tumor and healthy muscle tissues to evaluate signal intensity(expressed as Average Radiant Efficiency). The ratio between thefluorescence signal in the tumor and in the muscle (background tissue)was then calculated to assess the contrast.

The tumor-to-backgroud ratio of representative Compounds 1 and 4 isdisplayed in Table VI. These results show a remarkably high tumoruptake, suggesting tumor-specific accumulation.

TABLE VI Ex vivo tumor and excretory organs-to-muscle ratio 24 h afteradministration of the Compounds 1 and 4 in glioblastoma tumor bearingmice Tumor-to-background ratio (n = 5/group), (mean ± St. Dev) Compound1 6.92 ± 0.64 Compound 4 7.64 ± 0.86

Human Head and Neck Cancer

Tumor uptake experiments were performed in an animal model of human headand neck cancer (orthotopic), using Detroit-562 cells, overexpressing inparticular integrin receptor α_(v)β₆. Briefly, the human pharyngealcarcinoma cells Detroit-562 (ATCC, CCL-138) were cultured in Eagle'sMinimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum(FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin.Male Balb/c nu/nu mice, 4-6 weeks of age (Charles River Laboratories),underwent orthotopic implantation in the anterior portion of the tongueof about 2.5 million cells suspended in 0.03 mL di EMEM. Mice werehoused 4 per cage with food and water ad libitum. Animals were fed withVRF1 (P) sterile diet (Special Diets Services Ltd) up to the end of theacclimation period (5 days). Then, AIN-76a rodent diet irradiated(Research Diets), a special diet that reduces auto-fluorescence, wasused up to the end of the experiments. Tumor growth was monitored bylongitudinal assessments using a caliper up to the target size of 10-20mm³ (7-10 days after cell implantation).

Imaging experiments were performed using the preclinical optical systemIVIS Spectrum (Perkin Elmer). Animals were intravenously injected(lateral tail vein) with 3 nmol/mouse, at 24 hours post-administrationwere euthanized by anesthesia overdose, and the tongues were excised forex vivo optical imaging. Regions of interest (ROIs) were drawn on theanterior portion of the tongue (site of tumor cell implantation) and onthe posterior region (healthy tissue) to derive the tumor-to-backgroundratio.

Ex vivo imaging performed 24 h after the administration of the Compounds1 and 2 revealed a bright region in the tongue site of implantation ofthe tumor cells. Differently, the healthy region in the back of thetongue showed low signal, suggesting a low retention in healthy tissue.The administration of the compounds of the invention reveals thelocation of the tumor with moderate (TBR ˜2) tumor-to-backgroundcontrast, as shown in Table VII.

TABLE VII Ex vivo IVIS mean tumor-to-background ratio (TBR) 24 h afteradministration of Compunds 1 and 2 in H&N tumor bearing mice.Tumor-to-background ratio (n = 5/group), (mean ± St. Dev) Compound 12.27 ± 0.50 Compound 2 2.26 ± 0.10

Human Colorectal Cancer

Tumor uptake experiments were performed in an animal model of humancolorectal cancer (subcutaneous), using HT-29 cells, expressing lowlevels of integrin receptors. Briefly, the human colorectaladenocarcinoma cells HT-29 (ATCC, HTB-38) were cultured in McCoy's 5 Amedium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100IU/mL penicillin and 100 μg/mL streptomycin. Male Athymic nude mice, 4-6weeks of age (Envigo), underwent subcutaneous implantation (right flank)of about 5 million cells suspended in 0.1 mL of serum-free medium. Micewere housed 4 per cage with food and water ad libitum. Animals were fedwith VRF1 (P) sterile diet (Special Diets Services Ltd) up to the end ofthe acclimation period (5 days). Then, AIN-76a rodent diet irradiated(Research Diets), a special diet that reduces auto-fluorescence, wasused up to the end of the experiments. Tumor growth was monitored bylongitudinal assessments using a caliper up to the target size of300-600 mm³ (3-4 weeks after cell implantation). Imaging experimentswere performed using the preclinical optical system IVIS Spectrum(Perkin Elmer).

In vivo imaging was performed under gas anesthesia (Sevofluorane 6-8% inoxygen). Animals were intravenously injected (lateral tail vein) withthe compounds of interest, and euthanized after 24 hpost-administration. Regions of interest (ROIs) were drawn on theexcised tumor and healthy reference tissue (muscle) to evaluate signalintensity (expressed as Average Radiant Efficiency). The ratio betweenthe fluorescence signal in the tumor and in the muscle (backgroundtissue) was then calculated to assess the tumor-to-background ratio(TBR).

As shown in Table VIII, the representative Compounds 1 and 2 showedmoderate tumor-to-background ratio (TBR ˜4), allowing to clearlydelineate the tumor tissue from the healthy background.

TABLE VIII Ex vivo tumor-to-backround ratio (mean, SD, n = 5) 24 h afteradministration of Compounds 1 and 2 in colorectal cancer bearing mice.Tumor-to-background ratio (n = 5/group), (mean ± St. Dev) Compound 14.30 ± 1.50 Compound 2 4.20 ± 0.80

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The invention claimed is:
 1. A compound of formula (I),

wherein X is direct bond or —O—; Y is a group selected from linear orbranched C₁-C₆ alkyl, C₃-C₇ cycloalkyl and heterocyclyl, substituted byat least two hydroxyl groups; R1 and R2 are each independently a linearor branched C₁-C₆ alkyl substituted by a group selected from —SO₃H,—COOH, —CONH₂ and —COO—C₁-C₆ alkyl; and R3 is hydrogen, —SO₃H or alinear or branched C₁-C₆ alkyl substituted by —COOH or —CONH—Y, whereinY is a group selected from linear or branched C₁-C₆ alkyl, C₃-C₇cycloalkyl and heterocyclyl, substituted by at least two hydroxylgroups, or a stereoisomer or pharmaceutically acceptable salt thereof.2. The compound of formula (I) according to claim 1, wherein Y isselected from the group consisting of


3. The compound of formula (I) according to claim 1, which isrepresented by formula (Ia)

wherein X, R1, R2 and R3 are as defined in claim
 1. 4. The compound offormula (I) according to claim 3 which is selected from


5. A conjugate of a compound (I) as defined in claim 1 represented by acompound of formula (II)

wherein X is direct bond or —O—; Y is a group selected from linear orbranched C₁-C₆ alkyl, C₃-C₇ cycloalkyl and heterocyclyl, substituted byat least two hydroxyl groups; R1 is linear or branched C₁-C₆ alkylsubstituted by a group selected from —SO₃H, —COOH, —CONH₂ and —COO—C₁-C₆alkyl; R4 is linear or branched C₁-C₆ alkyl substituted by a groupselected from —SO₃H, —COOH and —CONH—(S)_(m)-T, wherein S is a spacer; Tis a targeting moiety; and m is an integer equal to 0 or 1; and R5 isselected from hydrogen, —SO₃H, a linear or branched C₁-C₆ alkylsubstituted by —COOH or —CONH—Y, and a group CONH—(S)_(m)-T, wherein Y,S, T and m are defined above; and wherein at least one between R4 and R5is linear or branched C₁-C₆ alkyl substituted by CONH—(S)_(m)-T, or astereoisomer or pharmaceutically acceptable salt thereof.
 6. Thecompound of formula (II) according to claim 5, wherein S is selectedfrom —(CH₂)_(p)COO—, —(CH₂CH₂O)_(p)CH₂CH₂COO— and—(CH₂CH₂O)_(p)CH₂CH₂NH—, wherein p is an integer between 0 and
 20. 7.The compound of formula (II) according to claim 5, wherein T istargeting moiety selected from the group consisting of a small molecule,a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibodyor fragment thereof and an aptamer.
 8. The compound of formula (II)according to claim 7, wherein T is a moiety interacting with an integrinreceptor.
 9. The compound of formula (II) according to claim 5,represented by the formula (IIa)

wherein R1, R4, R5 and X are as defined in claim
 5. 10. The compound offormula (II) according to claim 9 which is selected from


11. A compound as defined in claim 5 for use as fluorescent probes forbiomedical optical imaging applications in mammals.
 12. The compound foruse according to claim 11 wherein the imaging applications are directedto the detection of normal tissues and comprise angiography, perfusionimaging, bile duct imaging and nerve imaging.
 13. The compound for useaccording to claim 11, wherein the imaging applications are directed tothe detection of abnormal tissues and are carried out under NIRradiation.
 14. A pharmaceutical diagnostic composition comprising acompound as defined in claim 5 and at least one pharmaceuticallyacceptable carrier or excipient.
 15. Diagnostic kit comprising at leastone compound as defined in claim 5 together with additional adjuvantsthereof.
 16. The compound of formula (II) according to claim 6, whereinT is targeting moiety selected from the group consisting of a smallmolecule, a protein, a peptide, a peptidomimetic, an enzyme substrate,an antibody or fragment thereof and an aptamer.
 17. The compound offormula (II) according to claim 16, wherein T is a moiety interactingwith an integrin receptor.
 18. The compound of formula (II) according toclaim 6, represented by the formula (IIa)


19. The compound of formula (II) according to claim 7, represented bythe formula (IIa)


20. The compound of formula (II) according to claim 8, represented bythe formula (IIa)